STEM CELLS AND DEVELOPMENT 17:199205 (2008) Mary Ann Liebert, Inc. DOI: 10.1089/scd.2007.

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Original Research Report In Vitro Secreting Profile of Human Mesenchymal Stem Cells
TIMO SCHINKTHE,1 WILHELM BLOCH,2 and ANNETTE SCHMIDT2

ABSTRACT In addition to their multilineage potential, mesenchymal stem cells (MSCs) also have a wide range of functionality. Not only can MSCs reconstruct a tissue, but they also have the ability to control or cure other cells and can reconstruct a coordinating function. The opportunity to control other cells depends on MSCs being able to secrete factors like cytokines and chemokines. Therefore, we focused on asking, Which factors can be secreted by human MSCs? To answer this question, we analyzed the secreting profile of in vitro-expanded MSCs by using cytokine arrays. The media concentrations of 44 of the 120 analyzed cytokines were significantly increased by MSCs. Conversely, concentrations of 40 cytokines given with the sera were significantly decreased. The data presented here provide an overview about a large range of factors that were secreted by MSCs under cell culture conditions. These data indicate that MSCs demonstrate all previously described functions in cellular interactions without an external stimulus. The MSCs secreted angiogenic, immunosuppressive, anti-apoptotic, and proliferation-stimulating factors. INTRODUCTION

ESENCHYMAL STEM CELLS (MSCs) are progenitors of all connective tissue cells. In the adults of multiple vertebrate species, MSCs have been isolated from bone marrow (BM) and other tissues, expanded in culture, and differentiated into several tissue-forming cells such as bone, cartilage, fat, muscle, tendon, liver, kidney, heart, and even brain cells. [1,2]. Comparative studies have demonstrated that MSCs from different sources show comparable characteristics, even though the expression profile differs for a large scale of genes [3]. The most commonly used source for MSCs is the bone marrow. MSCs seem to be a useful tool for cellular therapy in cases of injured tissues. Different types of tissue engineering have been examined in the last years using MSCs. Besides their capability to differentiate into various kinds of cells, MSCs are able to rebuild, e.g., bone tissue [4,5], cartilage tissue [6], and vessels [7]. The reconstruction

of vessels, or neovascularization, is of prime importance in a wide range of diseases. One focus of interest is the cure of cardiac infarct [814]. MSCs are not only able to reconstruct a tissue; they also have the ability to control or cure for other cells and are able to reconstruct a coordinating function. Le Blanc and colleges demonstrated the potent immunosuppressive effect of MSCs in humans [15]. Other groups have also shown the immunosuppressive activity in vitro and in vivo [16]. This activity may be based on the capability of MSCs to reconstitute a functional hematopoietic microenvironment in vivo [17]. The opportunity to control other cells depends on the capability to secrete many factors, like cytokines and chemokines. In addition to the fact that MSCs are able to control neovascularization, we would expect that MSCs are able to secrete a large number of angiogenic factors. The capability to engraft hematopoiesis depends on the care of hematopoietic stem and progenitor cells. Immunosuppressive activity is also coupled with secre-

1Universidad 2Department

Nacional de Quilmes, Buenos Aires, Argentina. of Molecular and Cellular Sport Medicine, German Sport University, 50933 Cologne, Germany.

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SCHINKTHE ET AL. tion of cytokines, which also acts in an immunosuppressive manner. mRNA expression studies have indicated that MSCs may have the possibility of producing different kinds of soluble factors [1820]. Therefore, we focused on the question: Which factors can be secreted byMSCs?

RESULTS
MSCs from independent donors were cultured for 3 days. As a control, HSCs as well as the medium used for MSCs in absence of cells were cultured using same conditions. The supernatant of the stem cell culture as well as the medium control were analyzed using the RayBiotech cytokine array for analyzing 120 different cytokines and chemokines. By using computer-based densitometry, the relative intensity in relation to the range between positive and negative control was calculated (Fig. 1).

MATERIALS AND METHODS Cell culture and manipulation

MSCs of human origin were obtained from bone marrow aspirates as well as bone marrow from femur and hip head as described before [2124]. Before preparing Ficoll-PaqueTM PLUS density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden), bone marrow was filtered (mesh 70 m). The first change of medium, consisting of minimum essential medium ( -MEM), 20% (vol/vol) fetal calf serum (FCS), 200 M L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin, was prepared 2 days after culturing at 95% humidity and 5% CO2. Cells were used until passage 3. For every passage or experiment, cells were plated at 2,000 cells/cm2. Medium was changed twice a week. Hematopoietic stem cells (HSCs) were obtained from unused clinical surplus peripheral blood of stem cell-mobilized donors. The HSCs were isolated using the magnetic cell separation system (MACS) from Miltenyi Biotech (Bergisch Gladbach, Germany) and anti-CD34 according to the manufacturers guidelines. HSCs were always used fresh for experiments. Experimental cultivation of HSCs was done using the complete medium StemPro-34 SFM (Invitrogen, CA), which consists of STEMPRO-34 SFM and STEMPRO-34 Nutrient Supplement. For proliferation, stem cell factor (SCF), thrombopoietin (TPO), and granulocyte colony-stimulating factor (G-CSF; SigmaAldrich, MO) were added to a final concentration of 100 ng/ml. This study was approved by the local ethics committee and conforms to the Declaration of Helsinki.

Serum analysis of cytokines and chemokines

A cytokine/chemokine array kit (Ray Biotech Inc., Norcross, GA) was used to detect a panel of 120 secreted cytokines and chemokines. The manufacturers recommended protocol was used. For quantitative analysis of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and tumor necrosis factor (TNF), the BD Cytometric Bead Array Flex system (CBA-Flex system) from BD Biosciences (San Diego, CA) was used. The manufacturers recommended protocol was used.

Densitometry
For densitometry analysis, the gray scale values were measured using the computer-based system ImageJ Version 1.33 from the National Institutes of Health (NIH) [25]. All values were recalculated as relative intensity in relation to the range between positive and negative control.

FIG. 1. (A) A typical secretion profile of MSCs using the cytokine array membrane. The positive and negative controls as well as blank positions are placed at two opposite corners. For calculation of the relative intensity, the gray scale value between negative and positive controls was used as the given range. The MSC supernatant showed an increased content of MCP-1 compared to the control (B), whereas the concentration of TNF- is reduced in the supernatant of MSCs (C).

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MSC SECRETING PROFILE By comparing MSC samples with medium controls and also the supernatant of HSCs, a high number of cytokines were secreted specifically by MSCs. For example, TIMP-2, which shows a relative intensity of 1%, was not significantly influenced by HSCs (10%), but it was increased in the supernatant of MSCs up to a mean value of 62%. The factor angiopoetin-2 increased in the supernatant of MSCs up to a mean value of 47%, whereas the relative concentration in HSCs (1%) compared to control (2%) remained unchanged (Fig. 2A). In contrast, other factors were consumed by the cells and therefore decreased in the supernatant. An overview of the 10 factors that underwent the highest decrease in the supernatant is shown in Fig. 2B. By comparing all MSC samples with the controls, 44 of the 120 cytokines were secreted in a significant manner based on a significance level of p 0.05. In contrast, 40 of the tested cytokines showed a significant decrease in the supernatant of MSCs. The secreted cytokines can be pooled in different groups. First of all, different factors that are able to prevent the extrinsic apoptosis were secreted by MSCs. Factors such as soluble TRAIL R3,

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FIG. 2. (A) Overview of the 10 most increased cytokines in the supernatant of MSCs (black bars) compared to medium control (white bars) and HSC supernatant (gray bars). (B) Comparison of the changes in cytokine concentrations in the supernatant of MSCs (black bars) and HSCs (gray bars) of the 10 most decreased factors.

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SCHINKTHE ET AL. TRAIL R4, or FAS receptor are examples of these. This effect was supported by consuming pro-apoptotic factors like tumor necrosis factor- (TNF- or TNF- . The second group contained factors that are immunosuppressive or factors for which changes result in an immunosuppressive effect. These are factors like interleukin-12 p40 (IL-12 p40), Acrp30, or the soluble IL-2 receptor . This effect was also supported by decreasing inflammatory factors like IL-1 / , IL-2, or RANTES. A third group included factors that are able to induce cell proliferation, especially in hematopoietic cells. These are factors like G-CSF, fibroblast growth factor-4 (FGF-4), or FGF-9. Finally, factors that are able to interfere with angiogenesis were secreted by MSCs. These were factors like angiopoietin-2, bFGF, TIMP-1, TIMP-2, or VEGF (Table 1). In contrast, the HSCs demonstrated only four factors that had shown a significant increase in the cell supernatant. Those significantly increased were IL-16, FGF-7, Dtk, and CTACK. To verify the results of the cytokine array, we also performed quantitative analysis of selected factors on an enlarged group of MSC samples. Using the CBA-Flex system, we analyzed the concentration of VEGF, IL-8, and TNF in 10 independent MSC samples. After 3 days of cell culture, the MSCs produced a mean value of 5,330pg/ml of VEGF. The production of IL-8 was a mean value of 345pg/ml, whereas TNF was not produced by all of the tested samples (Fig. 3).

DISCUSSION
The results presented here are the first large-scale description of factors that are secreted by human (h) bone marrow-derived MSCs. These were obtained with hMSCs that were isolated as described by Pittenger et al. [1]. Pittenger described that MSCs expressed, e.g., IL-6R and TNF-RII on their cell surfaces [1]. We found the soluble form of both receptors in the supernatant of our cultures. Former studies had also analyzed the secreting potential of hMSCs by quantifying the mRNA [1820]. These studies had described that the mRNA of IL-11 is expressed in hMSCs, and we also detected in this mRNA supernatant. Data were not in agreement in the case of G-CSF [19,20], which we also found in the supernatant. Kim and colleges did not detect the mRNA for IL-7, which also conforms with our secreting data [20]. Liu and colleges described a large-scale expression profile of hMSCs derived from human cord blood [26]. He described 30 factors that were also tested by us. Twelve of these factors were secreted in cord blood MSCs as well as in our hMSCs (e.g., FGF-4, IL-8, TIMP1, TIMP-2, VEGF). Seven factors were not secreted in both studies (e.g., IL-4, IL-7, TNF- , TNF- ). FGF-7 was secreted in cord blood-derived MSCs but not in bone marrow-derived MSCs. Liu described a secretion of 10 factors that did not agree with our data (e.g., IL-6, GMCSF, IGFBP-1). For all of these 10 factors, we also ob-

TABLE 1. Secreted Acrp30, AgRP, Angiopoietin-2, bFGF, BTC, EGF-R, FAS, FGF-4, FGF-9, G-CSF, GITR, GITR-Ligand, GRO, HGF, ICAM-3, IGF-1SR, IGFBP3, IGFBP6, IL-2R , IL-6R, IL-8, IL-11, IL-12p40, IL-17, Lymphotaktin, MCP-1, MIF, MIP-1 , MIP-1 , MIP-3 , MSP , NT-4, Oncostatin M, Osteoprotegerin, PIGF, sgp130, sTNF RII, TIMP-1, TIMP-2, TRAIL R3, TRAIL R4, uPAR, VEGF, VEGF-D

OVERVIEW

OF

ALL ANALYZED CYTOKINES Non-significant Amphiregulin, Angiogenin, Axl, BDNF, BMP-4, NGF, CCL-28, CNTF, CTACK, Dtk, ENA-78, Eotaxin02, Eotaxin-3, FGF-6, FGF-7, Flt-3 Ligand, GDNF, GRO , HCC-4, ICAM-1, IGF-1, IL-1R4/ST2, IL-1R , IL-1RI, IL-6, IL-10, IL-12p70, IL-13, IL-15, I-TAC, Leptin, LIGHT, MCP-2, sTNF-RI, TECK, Thrombopoetin

Consumed BLC, BMP-6, CK 8-1, EGF, Eotaxin, FracTalkine, GCP-2, GM-CSF, I-309, IGFBP1, IGFBP2, IGFBP4, IFN , IL-1 , IL-1 , IL-2, IL-3, IL-4, IL-5, IL-7, IL-16, MCP-3, MCP-4, M-CSF, MDC, MIG, MIP-1 , MIP-3 , NAP-2, NT-3, PARC, PDGF-BB, RANTES, SCF, SDF-1, TARC, TGF- 1 TGF- 3, TNF- , TNF-

Cytokines that showed a significantly increased concentration within the supernatant of MSCs were grouped as secreted. Cytokines that showed a significantly decreased concentration were grouped as consumed. All cytokines that had not shown a significant change in concentration were sorted into the nonsignificant group.

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10000

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1 VEGF
sample #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 mean median range VEGF [pg/ml] 5977 7835 2317 2910 1811 4574 13996 7032 1927 4924 5330 4749 1657-14192

IL-8
IL-8 [pg/ml] 973 604 345 217 213 256 355 202 41 246 345 251 38-982

TNF
TNF [pg/ml] 1 0 1 0 2 1 2 0 0 2 1 1 0-2

FIG. 3. Quantitative analyses of VEGF, IL-8, and TNF in the supernatant of MSCs compared to control. The MSC supernatant of 10 different donors was analyzed.

tained a positive signal in the RayBiotech cytokine array. But in all of these cases, the signal in the medium control was equal or higher. We postulate that the signal results from a cross-reactivity with the fetal cytokines contained in the medium. In case of TNF- and TNF- , we obtained a strong signal in the medium control that was significantly decreased in the hMSC culture. By analyzing the supernatant as well as the medium control with a non-cross-reactive CBA-system for TNF, all samples were negative. Functional grouping of the secreted and consumed factors indicated four independent units. The hMSCs are acting by soluble factors as anti-apoptotic, immunosuppressive, proliferation enhancing, and modulator for angiogenesis. Six factors from the TNF family were changed by hMSCs. Osteoprotegerin, TNF-RII, TRAIL R3, and TRAIL R4 were secreted, whereas TNF- and TNF- were consumed by the hMSCs. In contrast to the TRAIL receptors TRAIL-R1 and TRAIL-R2, which contain a cytoplasmic death domain and signal apoptosis, the two secreted receptors TRAIL-R3 and TRAIL-R4 only act as decoy receptors. They lack a functional death domain and do not mediate apoptosis [27]. Osteoprotegerin is also a member of the TNF receptor superfamily and is known to be produced by bone marrow stromal cells. Be-

sides its normal function, osteoprotegerin also protects breast cancer cells from TRAIL-induced apoptosis [28]. It can also act as a soluble factor in the regulation of bone mass [29]. Zappia and colleges have shown that MSCs are able to reduce the serum level of interferon- (IFN- and TNF- produced by activated T cells [30]. Both factors were also reduced by the tested MSCs. The TNF- reduction can be explained by secretion of soluble TNF receptor II, which acts as an antagonist for TNF- and TNF- [31]. IL-12 p40, but not the subunit IL-12 p70, were secreted by the MSCs. Homodimers of IL-12 p40 act antagonistically to IL-12-mediated immune response. It is known that IL-12 p40 reduces the activity of T helper cells [32]. MSCs inhibited IL-2-induced lymphocytic proliferation [33]. This can be explained by the observed secretion of soluble IL-2 receptor . In contrast, the secreted sIL-6 receptor promotes the growth of hematopoietic progenitor cells [34]. Different factors, including the secreted IGFBP3, were postulated, with which stromal cells were able to support proliferation of hematopoietic progenitor cells in a cell contact-free co-cultivation [35]. There is last, but not least, a large number of factors that are associated with angiogenesis and were secreted by MSCs. These were the classical angiogenic factors like angiopoietin-2, bFGF, FGF-4, FGF-9, TIMP-1, TIMP-2, VEGF, or VEGF-D. But factors like G-CSF are also able to accelerate neovascularization [36]. These angiogenic factors also have impacts on hematopoietic cell differentiation and proliferation. For example, FGF-9 is able to stimulate thrombopoiesis in vivo [37], or FGF-4 induces cell proliferation in megacaryocytes (38). MSCs are one of the two most common stem cells found in the bone marrow. Therefore, we also compared the results with HSCs. Whereas the MSCs had shown the described broad range of secreted factors, in the case of HSCs, it was only possible to identify four significant secreted factors. These findings underlined the outstanding importance of MSCs for the bone marrow environment. In summary, the presented data provide an overview about a large range of factors that were secreted by MSCs under cell culture conditions. These data indicate that MSCs demonstrate all previously described functions in cellular interactions without an external stimulus. The cells secreted angiogenic, immunosuppressive, antiapoptotic, and proliferation-stimulating factors.

concentration [pg/ml]

ACKNOWLEDGMENTS
This work was supported by a grant from Frauenfrderung SpoHo. We thank Johanna Dinter, Jennifer Ick, and Anika Voss for excellent technical assistant and Rajesh Kumar for his editorial assistance with this manuscript.

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Address reprint requests to: Dr. Annette Schmidt Department of Molecular and Cellular Sport Medicine German Sport University Cologne Carl-Diem-Weg 6 50933 Cologne, Germany E-mail: a.schmidt@dshs-koeln.de Received for publication April 14, 2007; accepted after revision September 25, 2007.

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